The prognosis for the 60,000 Americans diagnosed each year with head and neck squamous cell carcinoma (HNSCC) is grim with only 50%-60% of patients surviving 5 years. Surgery, radiation and chemotherapy are debilitating. Immunotherapy shows promise but benefits only a minority of HNSCC patients. Thus, there is still an unmet medical need to better understand the disease and to generate more effective targeted therapeutics. Our exploratory study will address these two goals. Accumulating evidence indicates that the AHR is highly expressed and chronically active in many cancers, including HNSCC, in the absence of xenobiotics (and regardless of tumor etiology), by virtue of their production of endogenous AHR ligands including tryptophan- derived metabolites in the Kynurenine (Kyn) pathway. Formation of these ligands is dependent on IDO or TDO which themselves are AHR regulated, creating an AHR amplification loop. In negative feedback loops, AHR- regulated CYP1A1 and CYP1B1 degrade endogenous ligands and AHR-regulated AHR repressor (AHRR) suppresses AHR activity. We postulate that these elements of an ?AHR circuit? contribute to a steady-state of AHR activity which drives tumor aggression (e.g., cancer stem cell induction). Furthermore, we postulate that these oncometabolites contribute to immunosuppression by polarizing AHR+ macrophages towards immunosuppressive tumor-associated macrophages (TAMs), inducing granulocytic myeloid-derived suppressor cells (MDSC-Gs), and/or skewing AHR+ T cells towards regulatory T cells (Tregs). Therefore, our central hypothesis is that the AHR drives a self-sustaining AHR circuit that generates tryptophan metabolites which enhance tumor aggressiveness and suppress tumor immunity. This hypothesis will be tested in two specific aims: Aim 1: Determine the relative contribution of factors controlling steady-state AHR signaling and Kyn production in human and murine HNSCC lines. AHR, IDO, TDO, CYP1A1, CYP1B1, and AHRR will be systematically deleted and a postulated resetting of the AHR steady-state, along with its biological consequences, assessed. The potential for environmental ligands to reset the steady-state also will be determined. AHR circuit consequences of these perturbations will be used to advance a novel mathematical model that, ultimately, can be used to describe and predict AHR activity and its biological consequences. Aim 2: Determine at what level the AHR acts as an immune checkpoint regulator in HNSCC. The hypothesis that malignant cells producing AHR ligand(s), by virtue of the AHR circuit (Aim 1), generate immunosuppressive TAMs, MDSC-Gs, and/or Tregs will be tested using unique orthotopic models of murine HNSCC and AHRLysM conditional knockout mice. In a translational subaim, we will pinpoint the immunologic target (TAMs, MDSC-Gs, and/or Tregs) of our second generation, commercially licensed AHR inhibitor. These interdisciplinary, collaborative studies will shed light on the dynamics of AHR ligand production, the AHR?s contribution to HNSCC, and the AHR as a new immune checkpoint target for HNSCC treatment.